Anti-proliferative and pro-apoptotic effects of statins on human pulmonary artery smooth muscle cells

Omar Ali, Ellena J Growcott, Martin R Wilkins & John Wharton, Section on Experimental Medicine & Toxicology, Imperial College London, W12 ONN, UK.

Pulmonary arterial hypertension (PAH) is a progressive disease that is characterized by the aberrant regulation of distal pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis. Current treatments do not address all the pathological processes implicated in the pathogenesis of PAH and none are curative. 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have pleiotropic effects, independent of their lipid lowering properties (Bonetti et al., 2003) . Statins have been reported to reverse pulmonary hypertension in rats by increasing apoptosis (Nishimura et al., 2003) and preventing endothelial dysfunction (Murata et al. 2005). We therefore sought to establish whether lypophilic (simvastatin, atorvastatin, lovastatin, mevastatin and fluvastatin) and lypophobic (pravastatin) statins have anti-proliferative and pro-apoptotic effects on isolated distal human PASMCs.

PASMCs were derived from distal human pulmonary arteries (n=8 isolates) and used at passage 3 to 12. Anti-mitogenic effects were determined by measuring [methyl-3H]-thymidine uptake and cell proliferation, stimulated by platelet derived growth factor-BB (5 ng/ml) and 5% serum respectively. Apoptosis was assessed by measuring cytoplasmic histone-associated-DNA fragments and the release of endothen-1 (ET-1) and matrix metalloproteinase-9 (MMP-9) measured in conditioned culture medium by immunoassay. Intracellular signalling was explored using mevalonate (MVA), geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) and inhibitors of geranylgeranyl transferase (GGTI-2133) and farnesyl transferase (FTI-277).

Lypophilic statins, but not pravastatin, attenuated DNA synthesis and cell proliferation in a concentration-dependent manner (10-8 to 10-5 M). Differences were also observed in the inhibitory potency of lypophilic statins, For example, 10-6 M simvastatin exhibited a greater inhibitory effect on thymidine uptake (43.0±5.9 % inhibition, mean±SEM, n=5 isolates, P<0.001) compared to 10-6 M atorvastatin (8.8±2.2 % inhibition). Simvastatin and atorvastatin both increased DNA fragmentation over 48 hours and opposed the anti-apoptotic effects of transforming growth factor-β1 (TGF-β1, 2 ng/ml) and exogenous ET-1 (10-8 M). Statins, such as simvastatin and atorvastatin, also displayed a concentration-dependant inhibitory effect on endogenous ET-1 and MMP-9 production, induced by TGF-β1 and co-stimulation with tumour necrosis factor-α (10 ng/ml) and phorbol ester (10-7 M) respectively. The inhibitory effects were reversed by addition of either MVA (10-4 M) or GGPP (10-5 M), but not FPP, and were mimicked by GGTI-2133 (10-5 M) rather than FTI-277, consistent with signalling via geranyl-geranylated proteins. Interestingly, t he Rho kinase inhibitor Y27632 (10-5 M) also inhibited ET-1 release (60±10 % inhibition, P<0.001, n=6).

The in vitro data suggest that statins have direct anti-proliferative and pro-apoptotic effects on distal human PASMCs and also inhibit the production of ET-1 and MMP-9 in these cells. These pleiotropic effects may be significant in the chronic treatment of PAH patients with lypophilic statins such as simvastatin.

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